The mu opioid receptor, the opioid receptor most closely associated with both analgesia and behavioral reinforcement, is a member of the extensive family of G protein-coupled receptors. During the past year we have engaged in three series of investigations related to this receptor. The first one concluded a long-standing investigation into the kinetics of ligand binding to receptors expressed on living cultured Chinese hamster ovary (CHO) cells. Previous years work demonstrated that distortions in the kinetics occur due to the confinement of cells even within the thickness of a single monolayer of cells. This year, analyses of existing data were concluded. A new development was the successful simulation of the diffusion and binding of drugs down this intercellular gap. The simulations show that such distortions are indeed expected and that they increase with increasing expression level of receptors, with the increasing forward rate constant for binding of a ligand, and with increases in the thickness of the unstirred layer above the cells. Moreover, the simulations show that different kinetics can arise at different depths through even a monolayer of cells, a result experimentally confirmed using the optical sectioning capability of confocal microscopy. The second series was concerned with the observation, by us and by two other laboratories, that the count of receptors using labeled naloxone exceeds (by about 2- fold) the estimate by another antagonist, beta-funaltrexamine. After many refined trials it was found that the ratio of naloxone sites to beta-funaltrexamine sites was 1.29 ? 0.07 (se; n=29), a significant but small ratio that was enhanced by osmotic stress. We expected that this change would be accompanied by a change in the ratio of monomeric to dimeric receptors, as estimated by quantitative western blotting. Extensive experiments showed some changes in the unglycosylated vs. glycosylated protein, but the dimer was rarely if ever even seen. Part of our concern was whether the difference could be attributed to penetration of the ligand into the cells to label internalized receptors. To address this question, we performed saturation experiments with the hydrophilic peptide [3H]-CTAP. These results are less precise than those from high affinity ligands, but they showed that the vast majority of receptors on our CHO cells are on the plasma membrane. The third series of experiments, in collaboration with Dr. Chris Surratt (Duquesne University), tested various mutations in the mu opioid receptor in the 7th and 8th putative helical domains, specifically the "NPxxY" motif that is thought to be part of a molecular latch that activates the receptor. The tyrosine of this motif, Tyr-7.53, is thought to bond, by pi electron stacking, with the phenylalanine Phe-7.60. Therefore, several mutations were constructed to look for functional differences using the Xenopus oocyte expression system. We sought differences in effective dose, examined whether naloxone, an antagonist, might inhibit a constant activity, and whether the maximum response for peptides (DAMGO) versus alkaloid (normorphine) drugs differed. No naloxone response was seen, and possible differences in effective doses were blurred by the broad dispersion of the data. However, we did see a clear change in the peptide vs. alkaloid maximum responses in the mutant Y7.53N and possibly in Y7.53A. An unanticipated finding was that responses to both DAMGO and normorphine had a clear bimodal distribution for mutant Y7.53F, possibly reflecting variations in the G proteins that prefer to couple to this mutant receptor.